Raf-1 protein serine/threonine kinase functions as a critical shuttle enzyme that connects stimulation of growth factor receptors and protein kinase C at the membrane with activation of early growth response genes in the nucleus. Recently, we have shown that another member of the Raf family, B-Raf, is activated in PC12 cells after nerve growth factor (NGF) stimulation. Receptor coupling of Raf-1 and B-Raf activation is controlled by p21Ras. Growth factor stimulation of cells results in the increased phosphorylation and concomitant kinase activation of Raf-1 and B-Raf. Most recently, a negative regulatory phosphorylation of Raf-1 by A-kinase has been described. We have studied the effects of cAMP on the Ras-Raf mitogen-activated protein kinase (MAPK) signaling cascade and neuronal differentiation of PC12 cells. We investigated the effect of cAMP on activation of both Raf-1 and B-Raf, which is also expressed in PC12 cells. As in rat fibroblasts, activation of Raf-1 was strongly suppressed by cAMP. In contrast, cAMP did not inhibit NGF or epidermal growth factor (EGF)-induced activation of B-Raf (assayed by phosphorylation of Map/Erk kinase-1 (MEK1). Since B-Raf lacks a PKA phosphorylation site, this is consistent with cAMP inhibition of Raf-1 being mediated by PKA phosphorylation. The MEK kinase activity of B-Raf induced by NGF or EGF was at least tenfold greater than Raf-1, suggesting that B-Raf is primarily responsible for MAPK activation. Because B-Raf activation is not blocked, cAMP does not inhibit the Ras-Raf-MAPK cascade in PC12 cells. In contrast, cAMP can synergize with the Ras-Raf pathway to activate MAPK in this cell system.